Ca 125 tumor antigen function and uses thereof

ABSTRACT

The present invention is directed to modulators of CA 125 tumor antigen, a recombinant nucleic acids, vectors, host cells, pharmaceutical compositions, uses of the foregeoing for negatively modulating CA 125 tumor antigen in mammalian cell, preventing and treating a CA 125 tumor antigen associated disease in a mammal, as well as methods of prevention and treatment of such diseases and methods of negatively regulating CA 125 tumor antigen in a mammalian cell.

FIELD OF THE INVENTION

This invention relates to CA 125 tumor antigen. More specifically, itrelates to modulators of CA 125 tumor antigen and to their uses in thetreatment and prevention of diseases wherein CA 125 tumor antigen isoverexpressed.

BACKGROUND OF THE INVENTION

Ovarian cancer is one of the leading causes of death in women over 40.Although most patients respond to initial treatment, the majorityrelapses partially due to the appearance of chemo-resistant tumor cells.In order to improve therapy, it is essential to understand theunderlying mechanisms responsible for the occurrence of ovarian cancer.

CA125 Antigen is the Most Important Clinical Marker of Ovarian Cancer

CA125 tumor antigen is the most important clinical marker of ovariancancer as it is used to monitor response to chemotherapy. Rising orfalling blood levels of CA125 correlate with progression or regressionof the disease. CA125 antigen was first detected in the early 80's usingthe MAb OC125 which was raised against the human ovarian carcinoma cellline OV433 isolated from a patient with serous papillarycystadenocarcinoma (1). The specific reactivity of the OC125 Mab to avariety of human ovarian carcinoma cell lines and paraffin-embeddedovarian carcinoma tissues has led to the development of aradioimmunoassay to detect the CA125 antigen in serum from ovariancancer patients (2). Using this assay, rising or falling levels of CA125were shown to correlate with progression or regression of diseasedemonstrating that CA 125 levels correlate with clinical course of thedisease (2-4). It is currently employed as a predictor of clinicalrecurrence in ovarian cancer and to monitor response to chemotherapytreatment (5-8).

CA125 Biochemical Studies

Despite the widespread use of CA125 as a clinical marker of ovariancancer, the biochemical and molecular nature as well as the function ofthis antigen are poorly understood. Previous biochemical studiesdemonstrated that the CA125 epitope is carried on a large glycoproteinwith a M.W. in the range of 2×10⁵-10⁶ Da, while others reported thatCA125 consists of many subunits of 50-200 kDa (9-14). The study of Lloydet al. showed that CA125 is a high molecular weight glycoprotein havingproperties of a mucin-type molecule (15). In these studies however, adefinite consensus regarding the molecular nature of CA 125 could not beelaborated and no information about its function was provided. A partialcDNA encoding CA125 was recently identified as MUC16. The deduced aminoacid sequence proposed an extracellular domain composed of 9 tandemrepeats rich in serine, threonine and proline followed by a uniqueregion, a potential transmembrane domain and a short cytoplasmic tail.CA125 is expressed in more than 80% of epithelial ovarian cancer but isnot detectable in normal ovary tissues. However its role in the diseaseis unknown.

There is therefore a crucial need to identify therapeutic targets inorder to treat the disease.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a therapeutic targetthat satisfies the above mentioned need.

The present inventors developed a novel strategy to study the role ofproteins that could not be previously studied because the gene was notknown or not available. Using this strategy, they derived uniquemodulators of CA125 tumor antigen. The present inventors propose afunctional link between CA125 tumor antigen and the pathogenesis ofovarian cancer as well as other diseases where CA 125 tumor antigen isoverexpressed, a non exclusive list of which comprises endometriosis,cervical cancer, fallopian tube cancer, cancer of the uterus, prostatecancer and lung cancer. The inventors' results have lead to theidentification of CA 125 tumor antigen and CA 125 tumor antigen functionas novel therapeutic targets for the treatment and prevention of thesediseases in mammals.

Accordingly, an object of the present invention provides for a modulatorcapable of negatively modulating a CA 125 tumor antigen in a mammaliancell.

According to a prefered aspect of the present invention, the modulatornegatively modulates cell surface expression of CA 125 tumor antigen.Preferably, the modulator sequesters CA 125 tumor antigen or a fragmentthereof within an organelle of a mammalian cell, such as the endoplasmicreticulum, the trans-golgi, the golgi, the mitochondrion, the cytoplasmor any other cellular compartment.

According to another prefered aspect of the present invention, themodulator is a single-chain antibody that specifically binds to CA 125tumor antigen or a fragment thereof. The single-chain antibody ispreferably derived from the OC 125 monoclonal antibody or VK-8monoclonal antibody. The single-chain antibody preferably comprises afragment coded by at least one sequence of the group consisting of SEQ.ID NOS 1 to 6.

According to a second object of the present invention, there is provideda recombinant nucleic acid comprising at least one sequence selectedfrom the group consisting of SEQ ID NOS 1 to 6.

According to a third object of the present invention, there is provideda vector comprising a recombinant nucleic acid as defined by the presentinvention.

According to a fourth aspect of the present invention, there is provideda host cell comprising at least one element selected from the groupconsisting of a modulator as defined by the present invention, arecombinant nucleic acid as defined by the present invention and avector as defined by the present invention.

According to fifth object of the present invention, there is provided apharmaceutical composition comprising a pharmaceutically acceptablecarrier and at least one element selected from the group consisting of amodulator as defined by the present invention, a recombinant nucleicacid as defined by the present invention, a vector as defined by thepresent invention, and a host cell as defined by the present invention.

According to a sixth object of the present invention, there is provideda use of at least one element selected from the group consisting of amodulator as defined by the present invention, a recombinant nucleicacid as defined by the present invention and a vector as defined by thepresent invention for negatively modulating a CA 125 tumor antigen in amammalian cell.

According to a seventh object of the present invention, there isprovides a use of at least one element selected from the groupconsisting of a modulator as defined by the present invention, arecombinant nucleic acid as defined by the present invention, a vectoras defined by the present invention, a host cell as defined by thepresent invention and a pharmaceutical composition as defined by thepresent invention for preventing and treating a CA125-tumor-antigen-associated disease in a mammal.

According to an eighth object of the present invention, there isprovided a method of prevention or treatment of a CA125-tumor-antigen-associated disease in a mammal comprising the step ofadministrating to that mammal at least one element selected from thegroup consisting of a modulator as defined by the present invention, arecombinant nucleic acid as defined by the present invention, a vectoras defined by the present invention, and a host cell as defined by thepresent invention.

According to a ninth object of the present invention, there is provideda method for negatively modulating a CA 125 tumor antigen in a mammaliancell comprising the step of introducing into that cell at least oneelement selected from the group consisting of a modulator as defined bythe present invention, a recombinant nucleic acid as defined by thepresent invention, and a vector as defined by the present invention.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non restrictivedescription of preferred embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the proposed structure of the CA 125 tumor antigen.

FIGS. 2A and B show the construction of an ScFv library.

FIGS. 3A and B show the selection of soluble ScFvs through a “colonylift assay”.

FIG. 4 shows selection of soluble ScFvs (periplasmic extrcts)

FIG. 5 shows expression of ScFvs (ELISA).

FIG. 6 shows expression of ScFvs in the pCantab-5E prokaryoticexpression system with and without induction.

FIG. 7 shows selection of ScFvs binding to CA 125 (ELISA).

FIG. 8 shows selection of ScFvs binding to CA 125 (ELISA).

FIG. 9 shows cloning of ScFvs binding to CA 125 in eukaryotic expressionsystem.

FIGS. 10A, B, C and D show Western blots showing expression of ScFvsdirected to the golgi in OVCAR-3 and directed to the ER in OVCAR-3.

FIG. 11 shows expression of ScFv OC125 golgi 3.11 compared withexpression of CA 125.

FIG. 12 show expression of ScFv VK-8 KDEL 1.9 compared with expressionof CA 125.

FIG. 13 shows expression of control linker compared with expression ofCA 125.

FIG. 14 shows expression of ScFv in golgi and expression of ScFv in ERcompared with expression of proteins native to golgi and ER,respectively.

FIG. 15 illustrates construction and in vitro validation of anti-CA125scFvs.

A) CA125 binding activity of anti-CA125 OC125, VK-8-1.9 and VK-8-4.5scFvs present in periplasmic extracts of bacteria as well as anti-Bcl24D7 scFv compared to parental Mabs OC125 and VK-8 and to PBS andperiplasmic extract from bacteria, uninduced, IPTG-induced and controls.

B) Expression of scFvs from periplasmic extracts, probed with anti-Etagantibody.

C) Immunoprecipitation and co-immunoprecipitation of Golgi- andER-targeted OC125-3.11 scFv from transient transfection ofpSTCF.GOLGI-OC125-3.11 and pSTCF.KDEL-OC125-3.11 in NIH:OVCAR-3 humanovarian cancer cells using anti-c-myc, anti-CA125 Mabs OC125 and VK-8,western blot probed with anti-c-myc 9E10 antibody.

D) Immunoprecipitation and co-immunoprecipitation of Golgi- andER-targeted VK-8-1.9 scFv from transient transfection ofpSTCF-GOLGI-VK8-1.9 and pSTCF.KDEL-VK8-1.9 in NIH:OVCAR-3 human ovariancancer cells using anti-c-myc, anti-CA125 Mabs OC125 and VK-8, Westernblot probed with anti-c-myc 9E10 antibody.

E) Immunoprecipitation and co-immunoprecipitation of ER-targetedVK-8-4.5 scFv from transient transfection of pSTCF.KDEL-VK8-4.5 inNIH:OVCAR-3 human ovarian cancer cells using anti-c-myc, anti-CA125 MabsOC125 and VK-8, western blot prober with anti-c-myc 9E10 antibody.

FIG. 16 shows localization of anti-CA125 scFvs and CA125 cell surfacedown regulation.

A) NIH:OVCAR-3 cells were transiently transfected withpSTCF.Golgi-OC125-3.11 or pSTCF.KDEL-VK-8-1.9 constructs and 48 hrslater the cells were fixed in ice-cold methanol. Localization of scFvswas detected with the anti-c-myc A14 polyclonal antibody and comparedwith ER and Golgi residents using anti-calreticulin PA3-900 and anti-ADPribosylation factor MA3-060 monoclonal antibodies, respectively. Oregongreen anti-rabbit and texas red anti-Mouse secondary antibodies wereused.

B) NIH:OVCAR-3 cellswere transiently transfected withpSTCF.Golgi-OC125-3.11 or pSTCF.KDEL-VK-8-1.9 constructs and 48 hrslater the cells were fixed in ice-cold methanol. Expression of scFvs andCA125 was detected using the anti-c-myc A14 polyclonal antibody andanti-CA125 M11 monoclonal antibody. Oregon green anti-rabbit and Texasred anti-mouse secondary antibodies were used.

FIG. 17 shows Cell surface down modulation of CA125 in stableNIH:OVCAR-3 clones expressing the ER-VK-8-1.9 anti-CA125scFv andrelevant control.

A) Stable transfectants expressing the ER-targeted VK-8-1.9 and VK-8-4.5scFvs and parental cell line NIH:OVCAR-3 were fixed in ice-cold methanolgenerated and expression of CA125 at the cell surface and scFv wasassesed by immunofluorescence using anti-c-myc A14 polyclonal antibodyand anti-CA125 M11 monoclonal antibody, respectively. Oregon greenanti-rabbit and Texas red anti-mouse antibodies were used as secondaryantibodies.

B) CA125 expression in the stable trasnfectants was analysed by FACSusing anti-CA125 M11 monoclonal antibody and a Phyco-Erythrin-anti-mouseantibody and compared with parental cell line NIH:OVCAR-3; Black,OVCAR-3 levels of CA125 expression at cell surface; grey, CA125 levelsin stable transfectants.

FIG. 18 shows decreased CA125 cell surface expression influences theproliferation rate, cell-cell interaction and cell migration.

A) Growth curve of stable NIH:OVCAR-3 transfectants ER-VK-8-1.9#9(positive for CA125 binding) and ER-VK-8-4.5#12 (negative for CA125binding) compared to parental cells OVCAR-3. Cells were plated intriplicate in 96-well plate and cell proliferation was measured everyday with a XTT assay. Plot represents results from 3 independentexperiments

B) Cell aggregation assay. Cells were plated onto 0.6% agarose layer inbacterial dishes. Seventy-two hours later photomicrographs were taken(10× magnification) to visualize the presence of cell aggregates.

C) Wound healing assay. A wound was made using a 13 mm-wide razor bladein confluent cell monolayers and 20 mM hydroxy-urea was added to blockcell proliferation. Forty-eight hours later, the cells were fied inmethanol and stained with Giemsa and microphotographs were taken (1OXmagnification).

D) Tumorigenic assay. Ten millions NIH:OVCAR-3 transfectantsER-VK-8-1.9#9, ER-VK-8-4.5#12 and parental cells OVCAR-3 were inoculatedsubcutaneously in nude mice Tumors were allowed to grow for 6 weeksafter which tumorw were excised and tumor weight was measured andplotted for each transfectant.

FIG. 18.1 shows results of clonogenic assay.

Increasing amounts of each transfectant, VK-8 KDEU1:9#9 and KDEL/4:5#12,and the parental cell line NIH OVCAR-3 were seeded in 6-well plates andgrown in the absence or the presence of doxycycline. Fourteen dayslater, cells were stained with Giemsa and colonies were scored. Arrowsshow colonies and numbers correspond to the amount of cells initiallyseeded. Number of colony formed was plotted against increasing amountsof cells seeded (FIG. 18.1A). Plates were scanned to visualize the sizeof stained colonies (FIG. 18.1B-D).

FIG. 19

A) NEDO cDNA clone FLJ14303 encodes a part of CA125. Cos-7 cells(negative for CA125 by Western blot and ELISA) were transfected with anexpression vector encoding the cDNA from the NEDO clone FLJ14303.Reactivity of anti-CA125 OC125 and VK-8 antibodies with the expressionproduct of this cDNA was analysed by western blot and compared to CA125expression in OVCAR-3 cells as well as in mock-transfected Cos-7 cells.

B) Expression of the CA125 cytoplasmic tail fused to Gal4 DNA bindingdomain. The CA125 cytoplasmic tail was cloned in the pGBDU and pGAD forthe yeast two-hybrid system. The S. Cerevisiae strain PJ69-4a wastransformed with the pGBDU empty vector (EV) or with the vectorContaining CA125 cytoplasmic tail (Cyto). Three days after growth onappropriate media, proteins were extracted from the 2 transfectants orthe wt strain PJ69-4a, ran on 12.5% SDS-PAGE and transfered by westernblot on a PVDF membrane. The membrane was probed with anti-Gal4 DNAbinding domain antibody Gal-4-DBD RK5C1.

FIG. 20 shows cisplatin sensibility of stable NIH:OVCAR-3 clonesexpressing the ER-VK-8-1.9 anti-CA125scFv and relevant controls.

Cells were plated in triplicate in 96-well plates and exposed or not toincreasing concentrations of cisplatin. Fours days later, cellproliferation was measured with a XTT assay. Percentage of survival wasplotted against concentration of cisplatin. Curves represent resultsfrom 3 independent experiments. Red line represents 50% survival.

FIG. 20.1 shows the relative distribution of cells in each phase of thecell cycle for transfectants ERVK-8-1.9#9 and ERVK-8-4.5#12.

DNA content of non-synchronized cells was stained with propidium iodideat various time points for approximately 48 hours. FIGS. 20.1A1 to A4show the different relative profiles of cell cycle progression: graphs001 to 007 (ERVK-8-4.5#12) and 008 to 014(ERVK-8-1.9#9) at 0, 8,16, 24,32, 40 and 48 hrs. FIG. 20.1B is a table form of cell cycle progressionand shows the relative percentages of cells in each phase of the cellcycle as determined by FACS analysis.

FIG. 21 shows IC50 of cisplatin for the stable NIH:OVCAR-3 clonesexpressing the ER-VK-B-1.9 anti-CA125scFv and relevant controls.Inhibitory concentrations of cisplatin resulting in 50% survival ofcells were calculated from curves of graph in FIG. 20 (red line in FIG.20).

FIG. 22 shows expression of E-cadherin and avp5 integrin in NIH:OVCAR-3cells. NIH:OVCAR-3 were fixed in ice-cold methanol generated andexpression of E-cadherin and αvβ5 integrin at the cell surface wasassesed by immunofluorescence using E-cadherin clone 36 and anti-αvβ5integrin clone P1F6 antibody and Texas red labelled secondary anti-mouseantibody.

FIG. 23 shows expression of E-cadherin and scFv in NIH:OVCAR-3 stabletransfectant expressing the ER-VK-8-1.9 anti-CA125 scFv withoutinduction with doxycycline. Cells were grown on glass slides for 48 hrsand fixed in ice-cold methanol generated and expression of E-cadherinand scFv was assesed by immunofluorescence using E-cadherin clone 36 andanti-c-myc A14 antibody and Texas red or Oregon green-conjugatedsecondary anti-mouse and anti-rabbit antibodies.

FIG. 24 shows expression of E-cadherin and scFv in NIH:OVCAR-3 stabletransfectant expressing the ER-VK-8-1.9 anti-CA125 scFv when inducedwith doxycycline. Cells were grown in presence of doxycycline for 48 hrsand then fixed in ice-cold methanol generated and expression ofE-cadherin and scFv was assessed by immunofluorescence using E-cadherinclone 36 and anti-c-myc A14 antibody and Texas red or Oregongreen-conjugated secondary anti-mouse and anti-rabbit antibodies.

FIG. 25 shows expression of E-cadherin and scFv in NIH:OVCAR-3 stabletransfectant expressing the ER-VK-4.5 control scFv without inductionwith doxycycline. Cells were grown on glass slides for 48 hrs and fixedin ice-cold methanol generated and expression of E-cadherin and scFv wasassesed by immunofluorescence using E-cadherin clone 36 and anti-c-mycA14 antibody and Texas red or Oregon green-conjugated secondaryanti-mouse and anti-rabbit antibodies.

FIG. 26 shows expression of E-cadherin and scFv in NIH:OVCAR-3 stabletransfectant expressing the ER-VK4.5 control scFv when induced withdoxycycline. Cells were grown on glass slides in the presence ofdoxycycline for 48hrs and fixed in ice-cold methanol generated andexpression of E-cadherin and scFv was assesed by immunofluorescenceusing E-cadherin clone 36 and anti-c-myc A14 antibody and Texas red orOregon green-conjugated secondary anti-mouse and anti-rabbit antibodies.

FIG. 27 shows expression of αvβ5 integrin and scFv in NIH:OVCAR-3 stabletransfectant expressing the ER-VK-8-1.9 anti-CA125 scFv when induced ornot with doxycycline. Cells were grown in the absence or presence ofdoxycycline for 48 hrs and subsequently fixed in ice-cold methanolgenerated and expression of αvβ5 integrin and scFv was assesed byimmunofluorescence using anti-αvβ5 integrin clone P1F6, anti-c-myc 9E10antibody and Texas re or Oregron green labelled secondary antibodies.

FIG. 28 shows expression of αvβ5 integrin and scFv in NIH:OVCAR-3 stabletransfectant expressing the ER-VK-8-4.5 anti-CA125 scFv when induced ornot with doxycycline. Cells were grown in the absence or presence ofdoxycycline for 48 hrs and subsequently fixed in ice-cold methanolgenerated and expression of αvβ5 integrin and scFv was assesed byimmunofluorescence using anti-αvβ5 integrin clone P1F6, anti-c-myc 9E10antibody and Texas re or Oregron green labelled secondary antibodies.

FIG. 29 shows alignment of deduced amino acid sequence for anti-CA125scFvs VK-8-1.9 and OC125-3.11 as well as control scFv VK-8-4.5Nucleotidic sequences encoding the anti-CA125 scFvs and their controlwere determined from pCANTAB5E/scFv constructs using the scFv specificprimers S1 and S6 from the pCANTAB5 sequencing primer set (AmershamPharmacia Biotech, Piscataway, N.J.). Sequences were determined usingthe LI-COR automatic sequencing system (Bio S&T Inc., Lachine, QUE).Amino acid sequence was deduced from the nucleotidic sequences andaligned using the Alibee multiple alignment software available atwww.genebee.msu.su/services/malign reduced.html. The area in boxesrepresent consensus sequences of frameworks 1-4 of heavy and lightchain, asterisks correspond to differences between VK-8-1.9 andOC125-3.11 anti-CA125 scFvs whereas arrows identify differences betweenVK-8-1.9 and VK-8-4.5 scFvs.

FIG. 30 shows SEQ ID NO: 1.

The 5′ to 3′ coding sequence of the head-to-tail linkedV_(H)-linker-V_(L) portion of the single-chain antibody VK-8-1.9. Thenucleotide sequence of the linker is underlined.

FIG. 31 shows SEQ ID NO: 2.

The 5′ to 3′ coding sequence of the head-to-tail linkedV_(H)-linker-V_(L) portion of the single-chain antibody OC125-3.11. Thenucleotide sequence of the linker is underlined.

FIG. 32 shows SEQ ID NO: 3.

The 5′ to 3′ coding sequence of the V_(H) portion of the single-chainantibody VK-8-1.9.

FIG. 33 shows SEQ ID NO: 4.

The 5′ to 3′ coding sequence of the V_(L) portion of the single-chainantibody VK-8-1.9.

FIG. 34 shows SEQ ID NO: 5.

The 5′ to 3′ coding sequence of the V_(H) portion of the single-chainantibody OC125-3.11.

FIG. 35 shows SEQ ID NO: 6.

The 5′ to 3′ coding sequence of the V_(L) portion of the single-chainantibody OC125-3.11.

While the invention will be described in conjunction with exampleembodiments, it will be understood that it is not intended to limit thescope of the invention to such embodiments. On the contrary, it isintended to cover all alternatives, modifications and equivalents as maybe included as defined by the appended claims.

DESCRIPTION

The present invention is directed to modulators of CA 125 tumor antigen,recombinant nucleic acids, vectors, host cells, pharmaceuticalcompositions, and methhods of use of the foregeoing for negativelymodulating CA 125 tumor antigen in mammalian cell in order to preventand treat a CA 125 tumor antigen associated disease in a mammal.

Modulators of the Present Invention

The present invention is directed to modulators capable of negativelymodulating of CA 125 tumor antigen in a mammalian cell. Morespecifically, the modulators of the invention negatively modulate thefunction and or expression of CA 125 tumor antigen. Negative modulationis to be understood herein therefore as a significant decrease andpreferably the abolition of the function and or expression of CA 125tumor antigen. The present inventors show herein that CA 125 tumorantigen is responsible for the promotion of CA 125 associated diseasesin mammals. More particularly, the inventors have discovered that CA 125tumor antigen function and CA 125 tumor antigen expression bothconstitute novel therapeutic targets for combatting these diseases. Themodulators of the present invention are therefore aimed at these noveltherapeutic targets which are CA 125 tumor antigen expression and CA 125tumor antigen function.

The CA125 tumor antigen is a protein associated with the majority ofhuman epithelial ovarian cancers, the most common form of the disease.It is also known to be overexpressed in other diseases such asendometriosis, cervical cancer, cancer of the uterus, fallopian tubecancer, cancer of the endometrium, prostate cancer, lung cancer, etc.The foregoing diseases are therefore non exclusive examples of what ismeant by the expression “CA 125 tumor antigen associated diseases”.

As used herein, the term “mammal” refers to any mammal that issusceptible to a CA 125 tumor antigen associated disease as definedherein. Among the mammals which are known to be potentially affected,are humans.

More specifically, the present invention concerns a modulator capable ofnegatively modulating CA 125 tumor antigen function and or expression ina mammalian cell. Such a modulator may, for instance, negativelymodulate the cell surface expression of CA 125 tumor antigen. One way ofachieving this downregulation of CA 125 tumor antigen cell surfaceexpression is through the sequestration of newly synthesized CA 125tumor antigens or peptidic fragments thereof within cellularorganelle(s) such as the endoplasmic reticulum, the trans-golgi, thegolgi, the mitochondrion, the cytoplasm. The sequestration may alsoalternatively be achieved within any other cellular compartment.

A prefered modulator contemplated by the present invention is asingle-chain antibody that specifically binds to CA 125 tumor antigen ora peptidic fragment thereof. Such a single-chain antibody is preferablyderived from the OC 125 monoclonal antibody or the VK-8 monoclonalantibody as are the single-chain antibodies generated by the inventorsand designated hereinbelow OC 125-3.11 and VK-8-1.9. These particularsingle-chain antibodies contain fragments coded by SEQ ID Nos 2 and 1respectively (FIGS. 31 and 30). However, the invention also concerns anysingle-chain antibody comprising a fragment coded by any one or more ofthe sequences of SEQ. ID NOS 1 to 6 (FIGS. 30 to 35) and whichspecifically binds to CA 125 tumor antigen or a polypeptidic fragmentthereof and, as discussed herein, negatively modulates CA 125 tumorantigen function and or expression. Such an antibody preferably resultsfrom the combination, for instance, of the variable heavy sequencederived from the OC 125 monoclonal antibody (FIG. 5) linked to thevariable light sequence derived from the VK-8 monoclonal antibody (FIG.4). Or, preferably it results from the permutaion of the variable heavysequence derived from the VK-8 monoclonal antibody (FIG. 3) linked tothe variable light sequence derived from the OC 125 monoclonal antibody(FIG. 6). Other such permutations of SEQ ID Nos 1 to 6 are also withhinthe scope of the present invention.

The present inventors developed the two above mentioned anti-CA125single-chain antibodies (scFvs), OC 125-3.11 and VK-8-1.9, and show thatthey act as CA125-specific negative modulators of CA 125 tumor antigenfunction and expression as further described hereinbelow. When expressedintracellularly and retained to the ER (endoplasmic reticulum) or Golgi,the anti-CA125 scFvs entrap CA125 within the secretion pathway andtherefore prevent its proper cell surface localization in the mammaliancells which results in an increased cell proliferation and sensitivityto chemotherapeutic drugs such as cisplatin, reduced cell adhesion andmigration and prevents tumor growth in nude mice. The inventors show afunctional link between CA125 tumor antigen and cell proliferation,sensitivity to drugs such as cisplatin, cell adhesion and cell migrationand tumorigenicity.

Alternatively, the negative modulators of the present invention are tobe understood as comprising any negative modulator of CA 125 tumorantigen function and or expression. As people skilled in the art willknow, such negative modulators may act at different levels: such as thetransciptional, post-transcriptional, translational orpost-translational levels. Such negative modulators may be any type ofligand that specifically bind any CA 125 tumor antigen precursor (suchas a CA 125 mRNA), or any CA 125 tumor antigen, or fragments of theforegoing. Dominant negative molecules are another example of a negativemodulator which allows to achieve the negative modulation of CA 125tumor antigen function and or expression. A negative modulator of thepresent invention may therefore be any molecule which specificallyinhibits, blocks, neutralizes, knocks-out, or downregulates CA 125 tumorantigen function and or expression in a mammalian cell. Such a negativemodulator may also act upon CA 125 tumor antigen function or expressionfrom the outside of the cell, for instance, through the extracellularportion of CA 125 tumor antigen.

Recombinant Nucleic Acids

The present invention is also directed to any recombinant nucleic acidcomprising at least one nucleic acid sequence selected from the groupconsisting of SEQ ID NOS 1 to 6 (FIGS. 30 to 35 respectively).

The expression “nucleic acid sequence”, “nucleotide sequence”, “nucleicacid”, “polynucleotide”, “polynucleotide sequence” are terms which areemployed interchangeably in the present application and which are meantto designate a precise chain of nucleotides, modified or not,allowingthe defintion of a fragment or region of a polynucleotide, comprising ornot non natural nucleotides, and that may correspond to double-strandedDNA or a single-stranded DNA. This also includes DNA molecules, RNAmolecules, cDNA, artificial sequences and all fragments thereof. Anypolynucleotide which has been chemically, enzymatically, ormetabolically modified but which still retains the properties of theoriginal polynucleotide, e.g. codes for a peptide fragment whichspecifically binds to CA 125 tumor antigen in mammalian cells, isincluded within the meaning of the present invention.

The present invention does not concern nucleotide sequences in theirnatural chromosome environment, e.g. in the natural state. Rather itconcerns purified or isolated sequences, e.g. sequences that have beendirectly or indirectly obtained, through a process such as cloning,amplification and or chemical synthesis, their environment havingtherefore been at least partially modified. It is also understood that apolynucleotide which is introduced in an organism by transformation,genetic engineering, or any other recombinant method is “isolated” or“recombinant” even though it is present inside the said organism.

The sequences of the present invention preferably possess a DNA sequencepresenting a percentage of identity of 70% or more with one of thesequences of FIGS. 30 to 35. The expression “percentage of identity” ismeant to indicate a degree of identity between two nucleic acidsequences along the sequences in their entirety. If the particularsequences are of different lengths, the percentage of identity isexpressed relatively to the total length of the shortest sequence of thetwo. In order to calculate the percentage of identity, both sequencesare superposed in such a way to maximize the number of identical basesallowing for intervals, the number identical bases is is then divided bythe total number of bases of the shortest sequence.

As used herein, the term “polypeptide(s)” refers to any peptide orprotein comprising two or more amino acids joined to each other bypeptide bonds or modified peptide bonds. “Polypeptide(s)” refers to bothshort chains, commonly referred to as peptides, oligopeptides andoligomers and to longer chains generally referred to as proteins.“Polypeptide(s)” include those modified either by natural processes,such as processing and other post-translational modifications, but alsoby chemical modification techniques. Such modifications are welldescribed in basic texts and in more detailed monographs, as well as ina voluminous research literature, and they are well known to those ofskill in the art. It will be appreciated that the same type ofmodification may be present in the same or varying degree at severalsites in a given polypeptide. Also, a given polypeptide may contain manytypes of modifications. Modifications can occur anywhere in apolypeptide, including the peptide backbone, the amino acid side-chains,and the amino or carboxyl termini. Modifications include, for example,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, proteolytic processing,phosphorylation, prenylation, racemization, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation, selenoylation, sulfation and transfer-RNA mediatedaddition of amino acids to proteins, such as arginylation, andubiquitination. See, for instance: PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W.H. Freeman and Company, New York(1993); Wold, F., Posttranslational Protein Modifications: Perspectivesand Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OFPROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); Seifteret al., Meth. Enzymol. 182:626-646 (1990); and Rattan et al., ProteinSynthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad.Sci. 663: 48-62(1992). Polypeptides may be branched or cyclic, with orwithout branching. Cyclic, branched and branched circular polypeptidesmay result from post-translational natural processes and may be made byentirely synthetic methods, as well.

Vectors and Host Cells

In a third embodiment, the invention is further directed to cloning orexpression vector comprising a polynucleotide sequence as defined above,and more particularly directed to a cloning or expression vector whichis capable of directing expression of the polypeptide encoded by thepolynucleotide sequences of the present invention in a vector-containingcell or host cell.

As used herein, the term “vector” refers to a polynucleotide constructdesigned for transduction/transfection of one or more cell types.Vectors may be, for example, “cloning vectors” which are designed forisolation, propagation and replication of inserted nucleotides,“expression vectors” which are designed for expression of a nucleotidesequence in a host cell, or a “viral vector” which is designed to resultin the production of a recombinant virus or virus-like particle, or“shuttle vectors”, which comprise the attributes of more than one typeof vector.

A number of vectors suitable for stable transfection of cells andbacteria are available to the public (e.g. plasmids, adenoviruses,baculoviruses, yeast baculoviruses, plant viruses, adeno-associatedviruses, retroviruses, Herpes Simplex Viruses, Alphaviruses,Lentiviruses), as are methods for constructing such cell lines. It willbe understood that the present invention encompasses any type of vectorcomprising any of the polynucleotide molecule of the invention.

In a fourth embodiment, the invention is also directed to a host, suchas a genetically modified cell, comprising a modulator of the presentinvention, and or a vector of the present invention, and or any of thepolynucleotide sequences according to the invention and more preferably,a host capable of expressing the polypeptide encoded by thispolynucleotide.

The host cell is any type of cell. Preferably, it is a mammalian cell,such as a cell from an estblished cell line or an isolated primary cell.Alternatively, it may be an insect cell, yeast cell (Saccharomycescerevisiae, Ktuyveromyces lactis, Pichia pastoris), plant cell,microorganism, or a bacterium (such as E. coli).

Pharmaceutical Compositions

In a fifth embodiment, the present invention concerns any pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atleast one element selected from the group consisting of a modulator asdefined by the present invention, a recombinant nucleic acid as definedby the present invention, a vector as defined by the present invention,and a host cell as defined by the present invention.

Methods well-known in the art for making pharmaceutical compositions orformulations are found, for example, in “Remington's PharmaceuticalSciences” (Gennaro A R ed., 20th edition, 2000: Williams & Wilkins PA,USA). Formulations for parenteral administration may, for example,contain excipients, sterile water, or saline, polyalkylene glycols suchas polyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems include ethylene-vinylacetate copolymer particles, osmotic pumps, implantable infusionsystems, and liposomes. Formulations for inhalation may containexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or may be oily solutions for administration in theform of nasal drops, or as a gel.

Conventional pharmaceutical practice may be used to provide suitableformulations to administer the composition to patients. Administrationmay begin before the patient is symptomatic. Any appropriate route ofadministration may be employed, for example, administration may beparenteral, intravenous, intraarterial, subcutaneous, intramuscular,intracranial, intraorbital, ophthalmic, intraventricular, intracapsular,intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, bysuppositories, or oral administration. Therapeutic formulations may bein the form of liquid solutions or suspensions; for oral administration,formulations may be in the form of tablets or capsules; and forintranasal formulations, in the form of powders, nasal drops, oraerosols.

The amount of the elements in the composition of the present inventionand amounts to be administered is a therapeutically effective amount. Atherapeutically effective amount is that amount necessary for obtainingbeneficial results without causing overly negative secondary effects inthe host to which the composition is administered. The exact amount ofeach of the component elements in the composition and amount of thecomposition to be administered will vary according to factors such asthe type of condition being treated, the other ingredients in thecomposition, the mode of administration, the age and weight of theindividual, etc. If necessary, one may refer to the last edition of theCanadian Compendium of Pharmaceuticals & Specialties (CPS).

Uses and Methods

In a sixth embodiment, the present invention also concerns a use of atleast one element selected from the group consisting of a modulator asdefined by the present invention, a recombinant nucleic acid as definedby the present invention and a vector as defined by the presentinvention for negatively modulating a CA 125 tumor antigen in amammalian cell.

In a seventh embodiment, the present invention concerns a use of atleast one element selected from the group consisting of a modulator asdefined by the present invention, a recombinant nucleic acid as definedby the present invention, a vector as defined by the present invention,a host cell as defined by the present invention and a pharmaceuticalcomposition as defined by the present invention for preventing andtreating a CA 125-tumor-antigen-associated disease in a mammal.

In an eighth embodiment, the present invention concerns a method ofprevention or treatment of a CA 125-tumor-antigen-associated disease ina mammal comprising the step of administrating to that mammal at leastone element selected from the group consisting of a modulator as definedby the present invention, a recombinant nucleic acid as defined by thepresent invention, a vector as defined by the present invention, and ahost cell as defined by the present invention.

As used herein, the term “treating” refers to a process by which thesymptoms of a CA 125 tumor antigen associated disease are alleviated orcompletely eliminated. As used herein, the term “preventing” refers to aprocess by which a CA 125 tumor antigen associated disease is obstructedor delayed.

In a ninth embodiment, the present invention concerns a method fornegatively modulating a CA 125 tumor antigen in a mammalian cellcomprising the step of introducing into that cell at least one elementselected from the group consisting of a modulator as defined by thepresent invention, a recombinant nucleic acid as defined by the presentinvention, and a vector as defined by the present invention.

The present invention will be more readily understood by referring tothe following examples. Thess examples are illustrative of the widerange of applicability of the present invention and are not intended tolimit its scope. Modifications and variations can be made thereinwithout departing from the spirit and scope of the invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in practice for the testing of the present invention,the preferred methods and materials are described.

EXAMPLES Example I Construction and in vitro Validation of ModulatorsAccording to a Preferred Embodiment of the Present Invention, Namely,anti-CA125 scFvs

The inventors constructed single-chain antibody libraries derived fromthe OC125 and VK-8 hybridoma cell lines specific for CA125. The two scFvlibraries were screened for CA125 binding activity by ELISA usingcommercially purified human CA125. ScFvs that bound to CA125 by ELISA,OC125-3.11 and VK-8-1.9, and one that did not bind, VK-8-4.5 wereselected (FIG. 15A-B). The inventors hypothesized that if those scFvs(CA125 binders) once expressed intracellularly were localized to andretained within the ER or the Golgi then CA125 antigen would beentrapped during synthesis and thus be unable to localize at cellsurface and interact with other intracellular and/or extracellularproteins to achieve its function(s). The scFvs were targeted to the ERor trans-median Golgi by sequence fusion with an Igκ secretion leaderand a KDEL signal or fusion with the N-terminal 81 amino acids of humanbeta 1,4-galactosyltransferase, a protein resident of the trans-medialGolgi (47-49) in addition ot a c-myc tag at the C-terminus.Immunoprecipitation experiments showed that the anti-CA125 scFvs wereimmunoprecipitated with anti-c-myc antibody whereas only OC125-3.11 andVK-8-1.9 (both positive for CA125 binding) were co-immunoprecipitatedusing anti-CA125 OC125 and VK-8 MAbs (FIG. 5C-E). These resultsdemonstrate that the OC125-3.11 and VK-8-1.9 anti-CA125 scFvs bind toCA125 in vitro.

Example II Localization of anti-CA125 scFvs and Cell SurfaceDown-Regulation of CA125

Proper localization of the anti-CA125 scFvs of the invention intransient transfection of human ovarian cancer cells OVCAR-3 wasdemonstrated by immunofluorescence. Results obtained with ER-VK-8-1.9and GOLGI-OC125-3.11 are shown in FIG. 16A. Immunofluorescence studiesshowed that cells expressing the Golgi-targeted OC125-3.11 orER-targeted VK-8-1.9 lost expression of CA125 at the cell surface.However surrounding cells that did not express the scFvs (nottransfected) were positive for CA125 at the cell surface. In addition,the presence of the ER-targeted VK-8-4.5, which did not bind CA125 byELISA and immunoprecipitation experiments, did not affect expression ofCA125 in the cells expressing this scFv. These results show that theexpression and retention of ER- or GOLGI-targeted anti-CA125 scFvsresults in CA125 down-regulation at the cell surface. Anti-CA125 scFvsact therefore act as potent negative modulators of CA125.

Example III Consequences of CA125 Cell Surface Down-Regulation in HumanOvarian Cancer Cell Line NIH:OVCAR-3

To determine the effects of down-modulating CA125 expression at the cellsurface, the inventors derived stable clones encoding the ER-targetedVK-8-1.9 and Golgi-OC125-3.11 (both positive for CA125 binding) andER-VK-8-4.5 (negative for CA125 binding) scFvs in human ovarian cancercell lines OVCAR-3 (high expresser of CA125), OV-90 (moderate expresser)and SKOV3ip1 cells (low expresser). Some of the OVCAR-3 clones have beenalready characterized for scFv and CA125 expression and all of the otherclones (including in SKOV3ip1) have also been evaluated.Characterization of stable clones ER-VK-8-1.9#9 and ER-VK-8-4.5#12 isshown in FIG. 17. A dramatic decrease in CA125 expression at the cellsurface was observed in stable clone ER-VK-8-1.9#9 (positive for CA125binding) while CA125 expression was not affected in the cloneER-VK-8-4.5#12 (negative for CA125 binding) although the scFv in thisclone was expressed at adequate levels (FIG. 17A). Similar results wereobtained from FACS analysis (FIG. 17B). These results are consistentwith results obtained previously from transient transfectionexperiments. Taken together these results demonstrate that the scFvs ofthe present invention act as specific negative modulators of CA125 andthat the stable clones of the present invention provide unique tools.

Expression of E-Cadherin and αvβv Integrin at the Cell Surface

To further characterize the stable transfectants expressing theanti-CA125 scFvs the inventors evaluated the expression of E-cadherinand αvβv integrin at the cell surface for each stable transfectant.FIGS. 23 through 26 show that E-cadherin expression at the cell surfaceis not affected by the presence of the ER-VK-8-1.9 anti-CA125 scFv orthe control ER-VK-8-4.5 demonstrating that E-cadherin expression is notmodulated by CA125 levels. Expression of αvβv integrin at the cellsurface of ER-VK-8-4.5 transfectant is also not affected by theexpression of the scFv (FIG. 28). However, the stable transfectantexpressing the ER-VK-8-1.9 anti-CA125 scFv shows a reduced level of αvβvintegrin at the cell surface demonstrating that CA125 influences levelsof αvβv integrin expression at the cell surface.

Cell Proliferation on Adhesive Support

In vitro growth kinetics of ER-VK-8-1.9 and ER-VK-8-4.5 clones wasevaluated compared with that of the parental cell line using a XTT cellproliferation assay (50). Stable clone ER-VK-8-1.9#9 (positive for CA125binding) grew faster than the ER-VK-8-4.5#12 (negative for CA125binding) which grew at a rate similar to that of the parental OVCAR-3cells (FIG. 18A). Stable clone ER-VK-8-1.9#9 seems to adhere faster tothe plastic than OVCAR-3 cells or ER-VK-8-4.5#12 clone (not shown).These results show that loss of CA125 at the cell surface affects cellproliferation on adhesive support.

To further characterize this effect, clonogenic assays were performed toinvestigate the growth kinetics of each transfectant when seeded at lowdensity on adhesive support. Increasing amounts of each transfectant,VK-8 KDEL/1:9#9 and KDEL/4:5#12, and the parental cell line NIH OVCAR-3were seeded in 6-well plates and grown in the absence or the presence ofdoxycycline. Fourteen days later, cells were stained with Giemsa. Thenumber of colonies was scored for each transfectant and plotted againstthe amount of cells seeded. FIG. 18.1A shows that no difference in theamounts of colonies formed by each transfectant, regardless of CA125status, confirming that CA125 does not affect the ability of the cellsto form colonies when seeded at low density on adhesive support.However, FIGS. 18.1B to D show that the size of colonies formed bytransfectant VK-8-1.9#9 was much bigger and easier to visualize comparedto those from transfectant VK-8-4.5#12 and the parental cell line NIHOVCAR-3 demonstrating that transfectant VK-8-1.9#9 grow at an increasedrate compared to the controls. These results show that CA125 influencesthe growth rate of cells and does not however modulate the ability ofthe cells to form colonies on adhesive support when seeded at lowdensity.

Sensitivity to Cisplatin

Consequently, sensitivity to cisplatin of the various stabletransfectants was determined. Stable clone ER-VK-8-1.9#9, theER-VK-8-4.5#12 and the parental cell lines were plated in triplicate in96-well plates and exposed or not to increasing concentrations ofcisplatin. Fours days later, cell proliferation was measured with a XTTassay. Percentage of survival was plotted against concentration ofcisplatin. Curves represent results from 3 independent experiments (FIG.20). Results showed that stable clone ER-VK-8-1.9#9 was more sensitiveto cispaltin than control cells. IC50 were calculated and FIG. 21 showsthat the stable clone ER-VK-8-1.9#9 is approximately 10-fold moresensitive to cisplatin than ER-VK-8-4.5#12 and the parental cell lines.Similar experiments were performed using taxol and results showed nodifference between sensitivity of stable transfectant ER-VK-8-1.9#9, theER-VK-8-4.5#12 and the parental cell lines confirming that the increasedsensitivity of transfectant ER-VK-8-1.9#9 is linked to the increase incell proliferation. These results demonstrate that CA125 influences cellproliferation and thereby controls the sensitivity to therapeutics drugssuch as cisplatin.

Cell-Cycle Analysis

Cell proliferation is closely linked to rate of apoptosis and cellcycle. Therefore the cell cycle progression of each transfectant wasinvestigated. DNA content of non-synchronized cells was stained withpropidium iodide at various time points for approximately 48 hr and thepercentage of cells in each phase of the cell cycle was determined byFACS analysis. FIG. 20.1A shows that the respective profiles of cellcycle progression of transfectant VK-8-1.9#9 and VK-8-4.5#12 differsignificantly, mostly in the G2/M peak. FIG. 20.1B shows percentages ofcells in each phase of the cell cycle. A dramatic increase in the numberof cells in G2/M phase was obtained for transfectant VK-8-1.9#9 comparedto the control demonstrating that the loss of CA125 results in anincrease in cell cycle progression. These results therefore confirm thatCA125 controls cell proliferation by modulating the rate of cell cycleprogression.

Cell-Cell Interactions and Anchorage Independence

We also assessed the effect of CA125 cell surface down-regulation on theability of the cells to mediate cell-cell interaction using a cellaggregation assay (51). Cell-cell interactions are measured by theability of the cells to aggregate to each other and grow in clumps.Transfectant ER-VK-8-4.5#12 formed small aggregates and grew in smallclumps similarly to the parental OVCAR-3 cells (FIG. 18B). In contrast,transfectant ER-VK-8-1.9#9 (positive for CA125 binding) did not formaggregate and only isolated single cells were observed. In addition, thesingle cells observed in this clone did not grow and looked as if theywere dead or dying. These results show that loss of CA125 at the cellsurface impairs the cells ability to mediate cell-cell interactions andto survive in anchorage-independent conditions.

Cell Migration

The inventors also determined the consequence of reducing CA125expression levels at the cell surface on cell migration. We evaluatedthe cell motility of ER-VK-8-1.9#9 and ER-VK-8-4.5#12 stabletransfectants and compared to the parental cell line using the wound orscratch assay (52). Cells were plated in 6-well plates and whenconfluent a wound was made in the monolayer using a razor blade. Todistinguish between cell proliferation and cell migration, cellproliferation was inhibited with 20 mM hydroxyurea (53). Cells wereincubated in the presence or the absence of FBS. In the absence of FBSnone of the cells, neither the parental cells, were able to migrate andfill in the wound (not shown). This suggests that some factors presentin the serum may be required for stimulating cell motility as showed byothers in different tumor cell lines (54). However, in the presence ofFBS, the only cells that did not migrate and fill in the wound were thecells from clone ER-VK-8-1.9#9 (positive for CA125 binding) (FIG. 18C).Cells from clone ER-VK-8-4.5#12 (negative for CA125 binding) migrated ina similar manner as the parental cells. These results show that CA125affects cell migration of the OVCAR-3 cell line.

Tumor Growth

The inventors also determined whether the loss of CA125 expression atthe cell surface affects the in vivo behaviour of human ovarian cancercells in tumor-bearing mice subcutaneously or intraperitoneally. Thiswas achieved by evaluating tumor growth, tumor burden, formation ofascites, presence of tumor cells in ascites, pattern of metastasesspread and survival of mice. The tumorigenicity of each stabletransfectant was also determined in nude mice. Stable cloneER-VK-8-1.9#9, the ER-VK-8-4.5#12 and the parental cell lines wereinoculated subcutaneously in nude mice and tumors were allowed to growfor 6 weeks after which tumors were excised and tumor weight wasmeasured. FIG. 18D shows that tumor derived from stable cloneER-VK-8-1.9#9 were significantly much smaller (if existant) than thosefrom the ER-VK-8-4.5#12 and the parental cell lines demonstrating thatCA125 influence the tumorigenic potential of ovarian cancer cells. Wheninjected intraperitoneally, stable clone ER-VK-8-1.9#9 showed asignificant slower growth, reduced volume of ascites, a decrease in thetotal number of viable tumor cells in suspension (in ascites) andtherefore an overall increased in survival of mice (not shown).

These results taken together point to a role for CA125 in thepathogenesis of ovarian cancer by influencing tumor cell proliferation,tumor cell adhesion and migration, and in tumorigenesis. Results alsoshow CA 125 controls sensitivity of these cells to chemotherapeuticdrugs such as cisplatin.

Experimental Procedures

Derivation of anti-CA 125 scFv constructs—The hybridoma cell line VK-8which express a monoclonal antibody against CA 125 tumor antigen hasbeen described previously and was kindly provided by K. O. Lloyd(Sloan-Kettering Memorial Cancer Center, New York, N.Y.) (18). TotalmRNA was extracted from VK-8 hybridoma using the PolyA-rack kit fromPromega. Total mRNA extracted from OC125 hybridoma cell line was kindlyprovided by R. C. Bast (MD Anderson Cancer Center, Houston, Tex.). ScFvsconstructs were generated using the Recombinant Phage Antibody System(Amersham Pharmacia Biotech, Piscataway, N.J.) according to themanufacturer's instructions. Briefly, the variable heavy and lightchains (V_(H) and V_(L)) were amplified from the cDNA by PCR using mousevariable region primers. The V_(H) and V_(L) DNA fragments were linkedtogether by overlap extension PCR using a (Gly₄Ser)₃ linker to generate750 bp scFv constructs with flanking SfiI and NotI sites. The scFv DNAfragments were inserted into SfiI/NotI sites of the prokaryoticexpression vector pCANTAB5E from the Mouse ScFv Module (AmershamPharmacia Biotech, Piscataway, N.J.). Screening of recombinant clonesexpressing a soluble scFv was accomplished by a colony lift assay asdescribed previously (26).

Plasmids—The phagemid pCANTAB5E/scFv contains the anti-CA 125 scFvsencoding sequences under the control of the inducible lac promoter. ThescFvs are expressed as fusion proteins with a peptide tag (Etag) at theC-terminus to allow easy immunodetection. The ER-targeting vectorpSTCF.KDEL was previously described (27). The selected anti-CA 125 scFvDNA fragments were subcloned into the SfiI/NotI sites of pSTCF.KDEL justupstream and in frame with the c-myc tag/KDEL sequence. The pLTRretroviral expression vector was described in details elsewhere (28).The same anti-CA 125 scFv DNA fragments were subcloned into thepLTR.KDEL retroviral plasmid as SfiI/NotI sites after insertion of aSfiI/NotI containing polylinker at XhoI site of pLTR.KDEL. In thisvector, expression of the scFvs is under the control of atetracycline-inducible CMV promoter.

Binding analysis by ELISA—Periplasmic extracts were prepared aspreviously described (29). Elisa plates were coated with 10 μg ofpurified CA 125 tumor antigen in 200 μl carbonate buffer pH 9.6 andincubated overnight at 4°0 C. in a humidified chamber. Wells were washed3 times with 200 μl PBS and blocked with 200 μl 2% BSA/PBS-0.05% Tween20(for VK-8 derivative scFvs) or 200 μl 2% NFDM/PBS-0.1% Tween20 (forOC125 derivative scFvs) for 1 hr at room temperature in a humidifiedchamber. The plates were air dried, 100 μl of periplasmic extracts wereadded in 100 μl of blocking buffer and the plates were incubated at roomtemperature for 1 hr in humidified chamber. Parental monoclonalantibodies VK-8 and OC125 served as positive control and an anti-Bcl-2scFv and periplasmic extract from bacteria without plasmid served asnegative controls. The anti-Bcl-2 scFv was described previously (29).Plates were then washed 6 times with blocking buffer, wells were allowedto air dry, mouse anti-Etag antibody (Pharmacia Biotech, Piscataway,N.J.) was added 1:1000 in 200 μl 2% BSANPBS-0.05% Tween20 or 200□l 2%NFDM/PBS-0.1% Tween20 and plates were incubated for 1 h at 37° C. in ahumidified chamber. Wells were washed again 6 times with PBS/0.5%Tween20 or PBS/0.1% Tween20 and anti-mouse mouse HRP-conjugatedsecondary antibody 1:2000 was added in 2% BSA/PBS-0.05% Tween20 or 2%NFDM/PBS-0.1% Tween20 and plates were incubated for another hour at 37°C. in a humidified chamber. The plates were subsequently washed 10 timeswith PBS/0.05% Tween20 or PBS/0.1% Tween20, air dried, 100 μl of HRPsusbtrate were added and plates were incubated in darkness. After 15-30min 100 μl of H₂SO₄ 1N were added in each well to stop the colorimetricreaction. The OD was read at 450 ηm in an ELISA plate reader.

Sequencing of DNA encoding anti-CA 125 scFvs—ScFv encoded sequences weredetermined from pCANTAB5E/scFv constructs using the scFv specificprimers S1 and S6 from the pCANTAB5 sequencing primer set (AmershamPharmacia Biotech, Piscataway, N.J.). Sequences were determined usingthe LI-COR automatic sequencing system (Bio S&T Inc., Lachine, QUE).

Cell lines—The human ovarian cancer NIH:OVCAR-3 and the green monkeykidney COS-7 cell lines were obtained from the American Type CultureCollection (Rockville, Md.). COS-7 cells were maintained in F12/DMEM(Biomedia, Drummondville, QUE) supplemented with 10% FBS (Biomedia,Drummondville, QUE), 2 mM L-glutamine (Biomedia, Drummondville, QUE),100 units/ml penicillin (Cie, city, state) and 100 μg/ml streptomycin.NIH:OVCAR-3 cells were grown in RPMI 1640 (Biomedia, Drummondville, QUE)supplemented with 20% FBS (Biomedia, Drummondville, QUE), 2 mML-glutamine (Biomedia, Drummondville, QUE), 100 units/ml penicillin, 100μg/ml streptomycin and 10 μg/ml insulin. Both cell lines were maintainedat 37° C. in a humidified 5% CO₂ incubator.

Immunoblot analysis—Periplasmic extracts (equal volume), cell lysates(equal amounts of proteins) or immunoprecipitates were submitted toSDS-PAGE electrophoresis (12%) and transferred onto PVDF membrane. Themembranes were probed with either an anti-Etag antibody (AmershamPharmacia Biotech, Piscataway, N.J.) for periplasmic extracts or theanti-c-myc 9E10 antibody (Cie, city, state) for cell lysates. AHRP-conjugated rabbit anti-mouse antibody (Jackson ImmunoResearch, WestGrove, Pa.) was used at 1:10,000. The immunoblots were developed withchemiluminescence using commercially available ECL system according tothe manufacturer's instruction.

Immunoprecipitation—NIH-OVCAR-3 cells were transiently transfected withthe various scFv constructs using commercially available FuGene 6transfection agent using the manufacturer's instruction. Forty-eighthours later, cell were collected and lysed on ice in NP40 buffer (0.5%NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 mM NaCl, 1 mM EDTA, 20 mMTris-HCl pH 8.0). Protein concentration was measured by the Bradfordmethod using the Bio-Rad protein assay according to the instructionprovided by the manufacturer. Three hundreds micrograms of totalproteins were incubated with 1 ug of polyclonal anti-c-myc A-14 antibody(Santa Cruz), mouse anti-CA125 OC125 antibody or mouse anti-CA125 VK-8antibodies for 1 hr on ice without shaking. Thirty microliters of NP40buffer-prewashed protein A-sepharose beads (anti-c-myc) orprotein-agarose beads were added, slowly shaked for 1 hr at 4° C. andspun down at 5000 rpm for 5 min. The pellets were washed 3 times in NP40buffer and finally resuspended in 40 μl of NP40 buffer and boiled for 5min. Agarose or sepharose beads were pelleted and supernatants wereanalyzed by immunoblots for the presence of the various scFvs.

Stable cell lines expressing anti-CA 125 scFvs—The stable cell linesOVCAR3-1.9#9, OVCAR3-4.5#12 and OVCAR3-GFP were generated by stabletransfection of pLTR.KDEL-anti-CA 125 scFv#1.9, 2.7, 4.5 and GFPrespectively. Stable clones were selected under lug/ml blasticidinexposure. Stable transfection of NIH:OVCAR-3 cells was performed usingcommercially available FuGene6 transfection reagent followingmanufacturer's instructions.

Immunofluorescence—Transiently transfected NIH-OVCAR-3 cells or stableNIH:OVCAR-3 clones expressing the various anti-CA125 scFvs were grown onglass slide until a 50% confluence was reached. Glass slides were thenwashed in cold PBS and cells fixed in ice-cold methanol for 10 min at−20° C. Glass slides were next rinsed 5 min in cold PBS andpermeabilized in PBS containing 0.1% Triton X-100 for 5 min on ice andrinsed again in PBS. Slides were blocked in XmMPBS/2% BSA on ice for 45min and then incubated with primary antibodies in blocking buffer atroom temperature for 1 hr. Slides were next washed 3 times in cold PBS,incubated for 30 min at room temperature with anti-mouse or anti-rabbitsecondary antibodies coupled to Texas Red or Oregon green (1:1000),washed with PBS and mounted for visualization by fluorescencemicroscopy. Expression of the various anti-CA125 scFvs and CA125 wasdetected using anti-c-myc antibody (9E10, 1:500) and OC125 antibody(1:500). Localization of the scFvs was determined by comparing theirpattern of expression with that of calreticulin and ADP-ribosylationfactor for ER and Golgi localization using anti-calreticulin antibody(1:10000) anti-calreticulin and anti-ADP-ribosylation factor (1:400).

Flow cytometry analysis—Expression levels of CA125 and the anti-CA125scFvs in the various NIH:OVCAR-3 stable clones were analyzed by FACS.One million PBS/EDTA-treated cells were fixed in 2% para-formaldehydefor 20 min at room temperature. Cells were next permeabilized with 0.1%saponine for 20 min at room temperature and incubated with primaryantibodies in 2% BSA/PBS locking buffer for 45 min at room temperature.CA125 and scFv expression was determined using the mouse anti-CA125 M11antibody (1:500) and the polyclonal anti-c-myc antibody A14 (1:500)respectively. Cells were next pelleted and incubated with anti-mouse-PE(1:1000) and anti-rabbit-FITC (1:500) secondary antibodies in blockingbuffer for 45 min at room temperature. Cells were pelleted, resuspendedin an appropriate volume and analyzed using a FACS Scan cytometer(Becton Dickenson, Mississauga, Canada).

Although preferred embodiments of the present invention have beendescribed in detail herein and illustrated in the accompanying drawings,it is to be understood that the invention is not limited to theseprecise embodiments and that various changes and modifications may beeffected therein without departing from the scope or spirit of thepresent invention.

REFERENCES

1. Bast R C Jr, Feeney M, Lazarus H. Nadler L M, et al. Reactivity of amonoclonal antibody with human ovarian carcinoma. J Clin Invest 1981,68: 1331-1337.

2. Bast R C Jr, Klug T L, St-John E, Jenison E, et al. Aradioimmunoassay using a monoclonal antibody to monitor the course ofepithelial ovarian cancer. N Engl J Med 1983, 309: 883-887.

3. Canney P A, Moore M, Wilkinson P M and James R D. Ovarian cancerantigen CA125: a prospective clinical assessment of its role as a tumourmarker. Br J Cancer 1984, 50: 765-769.

4. Vergote I B, Bormer O P and Abeler V M. Evaluation of serum CA 125levels in the monitoring of ovarian cancer. Am J Obstet Gynecol 1987,157, 88-92.

5. Niloff, J M, Knapp R C, Lavin P T, Malkasian G D, Berek J S, MortelR, Whitney C, Zurawski V R and Bast R C Jr. The CA125 assay as apredictor of clinical recurrence in epithelial ovarian cancer. Am JObstet Gynecol 1986, 155: 56-60.

6. Fish R G, Shelley M D, Maughan T, Rocker I et al. The clinical valueof serum CA125 levels in ovarian cancer patients receiving platinumtherapy. Eur J Cancer & Clin Oncol 1987, 23: 831-835.

7. Makar A P, Kristensen G B, Bormer O P, Trope C G. Is serum CA 125 atthe time of relapse a prognostic indicator for further survivalprognosis in patients with ovarian cancer?Gynecol Oncol 1993, 49: 73-79.

8. Rustin G J S, Nelstrop A E, McClean P, Brady M F et al. Definingresponse of ovarian carcinoma to initial chemotherapy according to serumCA 125. J Clin Oncol 1996, 14: 1545-1551.

9. De Los Frailes M T, Stark S, Jaeger W, Hoerauf A et al. Purificationand characterization of the CA125 tumor-associated antigen from humanascites. Tumor Biol 1993, 14: 18-29.

10. Kobayashi H, Ida W, Terao T, Kawashima Y. Molecular characteristicsof the CA 125 antigen produced by human endometrial epithelial cells:comparison between eutopic and heterotopic epithelial cells. Am J ObstetGynecol 1993, 169: 725-730.

11. Matsuoka Y, Nakashima T, Endo K, Yoshida T, et al. Recognition ofovarian cancer antigen CA125 by murine monoclonal antibody produced byimmunization of lung cancer cells. Cancer Res 1987, 6335-6340.

12. Nagata A, Hirota N, Sakai T, Fujimoto M and Komoda T. Molecularnature and possible prsence of a mebranous glycan-phosphatidylinositolanchor of CA125 antigen. Tumor Biol 1991, 12: 279-286.

13. Yu Y H, Schlossman D M, Harrison C L, Rhinehardt-Clark A, Soper J T,Klug T L, Zurawski V R and Bast R C Jr. Coexpression of differentantigenic markers on moieties that bear CA 125 determinants. Cancer Res1991, 51: 468-475.

14. Zurawski V R Jr, Davis H M, Finkler N J, Harrison C L, et al. CancerRev 1988, 11-12: 102-118.

15. Lloyd K O, Yin B W T, Kudryashov V. Isolation and characterizationof ovarian cancer antigen CA125 using a new monoclonal antibody (VK-8):identification as a mucin-type molecule. Int J Cancer 1997, 71: 842-850.

16. Yin B W T. and Lloyd K O. Molecular cloning of the CA125 ovariancancer antigen. J. Biol. Chem. 2001 276: 27371-27375.

17. O'Brien T J, Beard J B, Underwood L J, Dennis R A, Santin A D andYork L. The CA125 gene: an extracellular superstructure dominated byrepeat sequences. Tumor Biol 2001, 22: 348-366.

18. O'Brien T J, Beard J B, Underwood L J and Shigemasa K. The CA125gene: a newly discovered extension of the glycosylated N-terminal domaindoubles the size of this extracellular superstructure. Tumor Biol 2002,23: 154-169.

19. Yin B W T, Dnistrian A. and Lloyd, K O. Ovarian cancer antigen CA125is encoded by the MUC16 mucin gene. Int. J. Cancer 2002, 98: 737-740.

20. Kabawat S E, Bast R C Jr, Welch W R, Knapp R C and Colvin R B.Tissue distribution of a coelomic-epithelium-related antigen recognizedby the monoclonal OC125. Am J Clin Pathol 1983, 79: 98-104.

21. Bischof P, Tseng L, Brioschii P A, Herman W L. Cancer antigen 125 isproduced by human endometrial stromal cells. Hum Reprod 1986, 1:423-426.

22. Karlan B Y, Amin W, Casper S E, Littlefield B A. Hormonal regulationof CA125 tumor marker expression in human ovarian carcinoma cells:inhibition by glucocorticoids. Cancer Res 1988, 48:3502-3506.

23. Brumsted J R, McBean J H, Deaton J L, Gibson M. CA-125 secretion byluteal phase endometrium in vitro. Hum Reprod 1990, 5: 682-684.

24. Marth C, Lang T, Kota A, Mayer I and Daxenbichier G. Transforminggrowth factor-beta and ovarian carcinoma cells: regulation ofproliferation and surface antigen expression. Cancer Lett 1990, 51:221-225.

25. Kurachi H, Adachi H, Morishige K, Adachi K, Takeda T, Homma H,Yamamoto T and Miyake A. Transforming growth factor-alpha promoterstumor markers secretion from human ovarian cancers in vitro. Cancer1996, 78: 1049-54.

26. Konishi I, Fendrick J L, Parmley T H, Quirk J G and O'Brien T J.Epidermal growth factor enhances secretion of the ovariantumor-associated cancer antigen CA125 from the human amnion WISH cellline. J Soc Gynecol Invest 1994, 1: 89-96.

27. Ishiwata I, Ishiwata C, Nazawa S, Ishiwata H. CA125 production bygynecologic tumors in vitro and its modulation induced by dibutyl cyclicadenosine monophosphate. Asia Oceania J Obstet Gynecol 1986, 12:285-290.

28. Bonfrer J M, Linders T C, Hageman P C, Hilkens J G, Korse C M andMolthoff C F M. Effect of paclitaxel (taxol) on CA125 expression andrelease by ovarian cancer cell lines. Tumor Biol 1997, 18: 232-240.

29. Marth C, Zeimet A G, Widschwendter M, Ludescher C, Kaern J, Tropé C,Gastl G, Daxenbichler G and Dapunt O. Paclitaxel and docetaxel-dependentactivation of CA125 expression in human ovarian carcinoma cells. CancerRes 1997, 57: 3818-3822.

30. Konishi I, Fendrick J L, Parmley T H, Quirk J G Jr and O'Brien T J.Epidermal growth factor enhances secretion of the ovariantumor-associated cancer antigen CA125 from the human amnion WISH cellline. J Soc Gynecol Invest 1993, 1: 89-96.

31. Fendrick J L, Konishi I, Parmley T H, Quirk J G and O'Brien T J.CA125 phosphorylation is associated with its secretion from the WISHhuman amnion cell line. Tumor Biol. 1997, 18: 278-289.

32. www.cbs.dtu.dk/cai-bin/

33. Marth C, Zeimeth A G, Widschwendter and Daxenbichler G. (1998)Regulation of CA125 expression in cultures human carcinoma cells. Int JBiol Markers 13(4): 207-209.

34. Auersperg N, Pan J, Grove B D, Peterson T, Fisher J, Maines-BandieraS, Somasiri S and Roskelley C D. E-cadherin inducesmesenchymal-to-epithelial transition in human ovarian surfaceepithelium. Proc. Natl. Acad. Sci. USA 1999, 96: 6249-6254.

35. Gendler S J and Spicer A P. Epithelial mucin genes. Annu RevPhysiol. 1995, 57: 607-634.

36. Cao Y, Blohm D, Ghadimi B M, Stosiek P, Xing P X and Karsten U.Mucins (MUC1 and MUC3) of gastrointestinal and breast epithelia revealdifferent and heterogenous tumr-associated aberrations in glycosylation.J Histochem Cytochem 1997, 45: 1547-57.

37. Hilkens J, Vos H L, Wesseling J, Boer M, Storm J, van der Valk S,Calafat J and Patriarca C. Is episialin/MUC1 involved in breast cancerprogression? Cancer Lett 1995, 90: 27-33.

38. Spicer A P, Rowse G J, Lindner T K, Gendler S J. Delayed mammarytumor progression in Muc-1 null mice. J Biol Chem 1995, 270:30090-30101.

39. Schroeder J A, Thompson M C, Mockensturm Gardner M, and Gendler S J.Transgenic MUC1 interacts with epidermal growth factor receptor andcorrelates with mitogen-activated protein kinase activation in the mousemammary gland. J Biol Chem 2001, 276: 13057-13064.

40. Pandey P, Kharbanda S and Kufe D. Association of the DF3/MUC1 breastcancer antigen with Grb2 and the SOS/Ras exchange protein. Cancer Res1995, 55: 4000-4003.

41. Suwa T, Honda Y, Makiguchi Y, Takahashi T, Itoh F, Adachi M,Hareyama M and Imai K. Increased invasiveness of MUC1 andcDNA-transfected human gastric cancer MKN74 cells. Int J Cancer 1998,76: 377-382.

42. Yamamoto M, Bharti L Y, Kufe D. Interaction of the DF3/MUC1 breastcarcinoma-associated antigen and beat-catenin in cell adhesion. J BiolChem 1997, 272: 12492-12494.

43. Bharti L Y, Chen D, Gong J and Kufe D. Interaction of GlycogenSynthase Kinase 3□ with the DF3/MUC1 carcinoma-associated antigen and□catenin. Mol Cell Biol 1998, 18: 7216-7224.

44. Regimbald L H, Pilarski L M, Longenecker B M, Reddish M A, ZimmermanG and Hugh J C. The breast mucin MUC1 as a novel adhesion ligand forendothelial intercellular adhesion molecule 1 in breast cancer. CancerRes 1996, 56: 4244-4249.

45. Kam J L, Regimbald L H, Hilgers J H, Hoffman P, Krantz M J,Longenecker B M and Hugh J C. MUC1 synthetic peptide inhibition ofintercellular adhesion molecule-1 and MUC1 binding requires six tandemrepeats. Cancer Res 1998, 58, 5577-5581.

46. Moniaux N, Escande F, Porchet N, Aubert J-P and Batra S. Structuralorganization and classification of the human mucin genes. Frontiers inbioscience 2001, 6: 1192-1206.

47. Llopis J, McCaffrey J M, Miyawaki A, Farquhar M G and Tsien R Y.Measurement of cytosolic, mitochondrial, and Golgi pH in single livingcells with green fluorescent proteins. Proc Natl Acad Sci USA 1998, 95:6803-6808.

48. Yamaguchi N and Fukuda M N. Golgi retention mechanism ofbeta-1,4-galactosyltransferase. Membrane-spanning domain-dependenthomodimerization and association with alpha- and beta-tubulins. J BiolChem 1995, 270: 12170-12176.

49. Gleeson P A, Teasdale R D and Burke J. Targeting of proteins to theGolgi apparatus. Glycoconjugate J 1994, 11: 381-394.

50. Hindshaw V S, Olsen C W, Dybdahl-Sissoko N and Evans D. Apoptosis: amechanism of cell killing by influenza A and B viruses. J Virol 1994,68: 3667-3673.

51. Boterberg T, Bracke M E, Bruyneel E A and Mareel M M. Cellaggreagation assays. In: Metastasis Research Protocols. Analysis of cellbehavior in vitro and in vivo. Ed. S A Brooks and U Schumacher. HumanaPress (2001) pp 33-45.

52. Niinaka Y, Haga A and Raz A. Quantification of cell motility. In:Metastasis Research Protocols. Analysis of cell behavior in vitro and invivo. Ed. S A Brooks and U Schumacher. Humana Press (2001) pp 55-60.

53. Nurugesan G, Sa G and Fox P L. High-density lipoprotein stimulatedendothelial cell movement by a mechanism distinct from basic fibroblastgrowth factor. Circ Res 1996, 74: 1149-1156.

54. Erdel M. Speiss E, Trifz G, Boxberger H J and Ebert W. Cellinteractions and motility in human lung tumor cell lines HS-24 and SB-3under the influence of extracellular matrix components and proteaseinhibitors. Anticancer Res 1992, 12: 349-360.

55. Elbashir S M, Harborth J, Lendeckel W, Yalcin A, Weber K and TuschlT. Duplexes of 21-nucleotide RNAs mediate RNA interference in culturesmammalian cells. Nature 2001, 411: 494-498.

56. Elbashir S M, Lendeckel W and Tuschl T. RNA interference is mediatedby 21- and 22-nucleotide RNAs. Genes and Dev 2001, 15: 188-200.

57. Elbashir S M, Martinez J, Patkaniowaks A, Lendeckel W and Tuschl T.Functional anatomy of siRNAs for mediating efficient RNAi in Drosophilamelanogaster embryo lysate. Embo J 2001, 20: 6877-6888.

58. Harborth J, Elbashir S M, Bechert K, Tuschl T and Weber K.Identification of essential genes in cultured mammalian cells usingsmall interfering RNAs. J Cell Sci 2001, 114: 4557-4565.

59. Paul C P, Good P D, Winer I and Engelke D R. Effective expression ofsmall interfering RNA in human cells. Nature Biotech 2001, 29: 505-508.

60. Miyagishi M and Taira K. U6-promoter-driven siRNAs with four uridine3′ overhangs efficiently suppress targeted gene expression in mammaliancells. Nature Biotech 2001, 29: 497-500.

61. Tuschl T. Expanding small RNA interference. Nature Biotech 2001, 20:446-448.

62. Provencher D M, Lounis H, Champoux L, Tétreault M, Manderson E, WangJ C, Eydoux P, Savoie R, Tonin P N and Mes-Masson A-M. Characterizationof four novel epithelial ovarian cancer cell lines. In Vitro Cell DevBiol 2002, 36: 357-361.

63. Hamilton T C. Young R C. Louie K G. Behrens B C. McKoy W M.Grotzinger K R. Ozols R F. Characterization of a xenograft model ofhuman ovarian carcinoma which produces ascites and intraabdominalcarcinomatosis in mice. Cancer Res 1984, 44:5286-90.

64. Yu D. Wolf J K. Scanlon M. Price J E. Hung M C. Enhancedc-erbB-2/neu expression in human ovarian cancer cells correlates withmore severe malignancy that can be suppressed by E1A. Cancer Res 1993,53:891-8.

65. Piché A, Grim J, Rancourt C, Gomez-Navarro J, Reed J C and Curiel DT. Modulation of Bcl-2 protein levels by an intracellular anti-Bcl-2single-chain antibody increases drug-induced cytotoxicity in the breastcancer cell line MCF-7. Cancer Res 1998, 58: 2134-2140.

66. Frankel A, Rosen K, Filmus J and Kerbel R S. Induction of anoikisand suppression of human ovarian tumor growth in vivo by down-regulationof Bcl-XL. Cancer Res 2001, 61: 4837-4841.

67. Grossman J. Molecular mechanisms of “detachment-inducedapoptosis-Anoikis”. Apoptosis 2002, 7: 247-260.

68. Bank H L. Rapid assessment of islet viability with acridine orangeand propidium iodide. In Vitro Cell Dev Biol 1988, 24: 266-273.

69. Kudoh M, Knee D A, Takayama S and Reed J C. Bag1 proteins regulategrowth and survival of ZR-75-1 human breast cancer cells. Cancer Res2002, 62: 1904-1909.

70. Cannistra S A, Kansas G S. Niloff J. DeFranzo B. Kim Y. OttensmeierC. Binding of ovarian cancer cells to peritoneal mesothelium in vitro ispartly mediated by CD44H. Cancer Res 1993, 53: 3830-3838.

71. Strobel T and Cannistra S A. Beta1-integrins partly mediate bindingof ovarian cancer cells to peritoneal mesothelium in vitro. GynecolOncol 1999, 73: 362-367.

72. Carreiras F. Denoux Y. Staedel C. Lehmann M. Sichel F. Gauduchon P.Expression and localization of alpha v integrins and their ligandvitronectin in normal ovarian epithelium and in ovarian carcinoma.Gynecol Oncol 1996, 62: 260-267.

73. Wilson K E. Bartlett J M. Miller E P. Smyth J F. Mullen P. Miller WR. Langdon S P. Regulation and function of the extracellular matrixprotein tenascin-C in ovarian cancer cell lines. Br J Cancer 1999, 80:685-692.

74. Erickson C A. Cell migration in the embryo and adult organism. CurrOpin Cell Biol 1990, 2: 67-74.

75. Stoker M and Gherardi E. Regulation of cell movement: the motogerniccytokines. Biochem Biophys Acta 1991, 1072: 81-102.

76. Liotta L A, Mandler R, Murano Gm Katz D A, Gordon R K, Chiang P Kand Schiffman E. Tumor cell autocrine factor. Proc Natl Acad Sci USA1986, 83: 3302-3306.

77. Scotton C J, Wilson J L, Milliken D, Stamp G and Balkwill F R.Epithelial cancer cell migration: a role for chemokine receptors? CancerRes 2001, 61: 4961-4965.

78. Venkatakrishnan G, Salgia R and Groopman J E. Chemokine receptorsCXCR-1/2 activate mitogen-activated protein kinase via the epidermalgrowth factor receptor in ovarian cancer cells. J Biol Chem 2000, 275:6868-6875.

79. Rosano L, Varmi M, Salani D, DiCastro V, Spinella F, Natali P G andBagnato A. Endothelin-1 induces tumor ptoeinase activation andinvasiveness of ovarian carcinoma cells. Cancer Res 2001, 61: 8340-8346.

80. Ellerbroek S M, Halbleib J M, Benavidez M, Warmka J K, Wattenberg EV, Stack M S and Hudson L. Phosphatidylinositol 3-kinase activity inepidermal growth factor-stimulated matrix metalloproteinase-9 productionand cell surface association. Cancer Res 2001, 61: 1855-1861.

81. Lu J, Xiao Y, Baudhuin L M, Hong G and Xu Y. Role of ether-kinkedlysophosphatidic acids in ovarian cancer cells. J Lipid Res 2002, 43:463-476.

82. Bourguignon L Y W, Zhu H, Shao L and Chen Y-W. CD44 interaction withc-Src kinase promotes cortactin-mediated cytoskeleton function andhyaluronic acid-dependent ovarian tumor cell migration. J Biol Chem2001, 276: 7327-7336.

83. Naylor M S, Stamp G W H, Davies B and Balkwill F R. Expression andactivity of MMPs and their regulators in ovarian cancers. Int J Cancer1994, 58: 50-56.

84. Niebdala M J, Crickard K and Bernacki R J. In vitro degradation ofextracellular matrix by human ovarian cancer cells. Clin Exp Metastasis1987, 181-197.

85. Stack M S, Ellerbroeck S M and Fishman D A. The role of proteolyticenzyme in the pathology of epithelial ovarian carcinoma. Int J Cancer1998, 12: 569-576.

86. Afzal S, Lalani E N, Foulkes W D, Boyce B, Tickle S, Cardillo M R,Baker T, Pignatelli M and Stamp G W H. Matrix metalloproteinase-2 andtissue inhibitor of metalloproteinase-2 expression and synthetic matrixmetalloproteinase-2 inhibitor binding in ovarian carcinomas and tumorcell lines. Lab Invest 1996, 74: 406-421.

87. Sternlicht M D, Bissel M J and Werb Z. The matrix metalloproteinasestromelysin-1 acts as a natural mammary tumor promoter. Oncogene 2000,19: 1102-1113.

88. Wilson C L and Matrisian L M. Matrilysin: an epithelial matrixmetalloproteinase with potentially novel functions. Int J Biochem 1996,28: 123-136.

89. Pendas A M, Uria J A, Jimenez M G, Balbin M, Freije J P andLopez-Otin C. An overview of collagenase-3 expression in malignanttumors and analysis of its potential value as a taget in antitumortherapies. Clin Chim Acta 2000, 291; 137-155.

90. Chambers S K, Gertz R E, Ivins C M and Kacinski B M. Thesignificance of urokinase-type plasminogen activator, its inhibition,and its receptor in ascites of patiens with epithelial ovarian cancer.Cancer (Phila.) 1995, 75: 1627-1633.

91. Ellerbroek S M, Fishman D A, Kearns A S, Bafetti L M and Stack M S.Ovarian carcinoma cell regulation of matrix metalloproteinase-2 andmembrane-type 1 matrix metalloproteinase through □1 integrin. Cancer Res1999, 59: 1635-1641.

92. Fishman D A, Bafetti L M and Stack M S. Membrane-typemetalloproteinase expression and matrix metalloproteinase-2 activationin primary human ovarian epithelial carcinoma cells. Invasion Metastasis1996, 16: 150-159.

93. Leber T M and Balkwill F R. Zymography: a single-step stainingmethod for quantitation of proteolytic activity on substrate gels.Analytical Biochem 1997, 249: 24-28.

94. Shibata K. Kikkawa F. Nawa A. Tamakoshi K. Suganuma N. Tomoda Y.Increased matrix metalloproteinase-9 activity in human ovarian cancercells cultured with conditioned medium from human peritoneal tissue.Clin Exp Metastasis 1997, 15: 612-619.

95. Cruet S, Carreiras F, Staedel C and Guaduchon P. Moléculesd'adhérence et proteases dans les cancer épithéliaux de l'ovaire.Médecine/Science 1999, 15: 645-654.

96. Yu J S, Sena-Esteves M, Paulus W, Breakefidle So and Reeves S A.Retroviral delivery and tetracycline-dependent expression ofIL-1B-converting enzyme (ICE) in a rat glioma model provides controlledinduction of apoptotic death in tumor cells. Cancer Res 1996, 56:5423-5427.

97. Dimri G P, Lee X, Basile G, Agosta M, Scott G, Roskelley C, MedranoE E, Linsken M, Rubelj I, et al. A biomarker that identifies senescenthuman cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA1995, 92: 9363-9367.

98.Ozols R, Rubin, S C, Thomas G and Robboy S. Epihtelial ovariancancer. In: Principles and practice of gynecologic oncology,Lippincott-Raven, 3^(rd) ed., pp. 981-1057, 2000.

99. James P, Halladay J and Craig E A. Genomic libraries and a hoststrain designed for highly efficient two-hybrid selection in yeast.Genetics 1996, 144: 1425-1436.

100. Tremblay A, Lamontagne B, Catala M, Yeung Y, Larose S, Good L andAbou Elela S. (2002) A physical interaction between Gar 1p and Rnt 1p isrequired for the nuclear import of H/ACA small RNA-asociated proteins.Mol Cell Biol 22: 4792-4802.

1. A modulator capable of negatively modulating a CA 125 tumor antigenin a mammalian cell.
 2. The modulator according to claim 1, wherein itnegatively modulates cell surface expression of CA 125 tumor antigen. 3.The modulator according to claim 1 or 2, wherein it sequesters CA 125tumor antigen or a fragment thereof within an organelle of a mammaliancell.
 4. The modulator according to claim 3, wherein the organelle isselected from the group consisting of the endoplasmic reticulum, thetrans-golgi, the golgi, the mitochondrion, the cytoplasm and a cellularcompartment.
 5. The modulator according to any one of claims 1 to 4,wherein it is a single-chain antibody that specifically binds to CA 125tumor antigen or a fragment thereof.
 6. The modulator according to claim5, wherein the single-chain antibody is derived from the groupconsisting of OC 125 monoclonal antibody and VK-8 monoclonal antibody.7. The modulator according to claim 5 or 6, wherein the single-chainantibody comprises a fragment coded by at least one sequence of thegroup consisting of SEQ. ID NOS 1 to
 6. 8. The modulator according toclaim 7, wherein the single-chain antibody is coded by a sequenceselected from the group consisting of SEQ. ID NOS 1 and
 2. 9. Arecombinant nucleic acid comprising at least one sequence selected fromthe group consisting of SEQ ID NOS 1 to
 6. 10. A vector comprising arecombinant nucleic acid according to claim
 10. 11. A host cellcomprising at least one element selected from the group consisting of: amodulator according to any one of claims 1 to 8; a recombinant nucleicacid according to claim 9; and a vector according to claim
 10. 12. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and at least one element selected from the group consisting of:a modulator according to any one of claims 1 to 8; a recombinant nucleicacid according to claim 9; a vector according to claim 10; and a hostcell according to claim
 11. 13. Use of at least one element selectedfrom the group consisting of: a modulator according to any one of claims1 to 8; a recombinant nucleic acid according to claim 9; and a vectoraccording to claim 10; for negatively modulating a CA 125 tumor antigenin a mammalian cell.
 14. Use of at least one element selected from thegroup consisting of: a modulator according to any one of claims 1 to 8;a recombinant nucleic acid according to claim 9; a vector according toclaim 10; a host cell according to claim 11; and a pharmaceuticalcomposition according to claim 12; for preventing and treating a CA125-tumor-antigen-associated disease in a mammal.
 15. A method ofprevention or treatment of a CA 125-tumor-antigen-associated disease ina mammal comprising the step of administrating to said mammal at leastone element selected from the group consisting of: a modulator accordingto any one of claims 1 to 9; a recombinant nucleic acid according toclaim 10; a vector according to claim 11; and a host cell according toclaim
 12. 16. A method for negatively modulating a CA 125 tumor antigenin a mammalian cell comprising the step of introducing into said cell atleast one element selected from the group consisting of: a modulatoraccording to any one of claims 1 to 9; a recombinant nucleic acidaccording to claim 10; and a vector according to claim 11.